CN109963864B - Alkaline stable FC binding proteins for affinity chromatography - Google Patents

Abstract

The present invention relates to Fc-binding proteins comprising one or more Fc-binding domains, wherein at least one domain comprises an amino acid sequence selected from SEQ ID NO: 1-6 or 21. The invention also relates to an affinity matrix comprising an Fc-binding protein of the invention. The invention also relates to the use of these Fc-binding proteins or affinity matrices for affinity purification of immunoglobulins and to methods of affinity purification using the Fc-binding proteins of the invention.

Description

Alkaline Stable FC-Binding Proteins for Affinity Chromatography

Field of invention

The present invention relates to Fc-binding proteins comprising one or more Fc-binding domains, wherein at least one domain comprises an amino acid sequence selected from SEQ ID NO: 1-6 or 21. The invention also relates to an affinity matrix comprising an Fc-binding protein of the invention. The invention also relates to the use of these Fc-binding proteins or affinity matrices for affinity purification of immunoglobulins and to methods of affinity purification using the Fc-binding proteins of the invention.

Background of the invention

Many biotechnological and pharmaceutical applications require the removal of contaminants from samples containing antibodies. An established method for capturing and purifying antibodies is affinity chromatography using bacterial cell surface protein A from Staphylococcus aureus as a selective ligand for immunoglobulins (see, e.g., Huse et al., J. Biochem. Biophys. Methods 51, 2002: 217-231). Wild-type protein A binds to the Fc region of IgG molecules with high affinity and selectivity and is stable at high temperatures and over a wide range of pH values. There are variants of Protein A with improved properties such as alkaline stability available for purifying antibodies, and a variety of chromatography matrices containing Protein A ligands are commercially available. However, chromatography matrices based in particular on wild-type protein A show a loss of binding capacity for immunoglobulins upon exposure to alkaline conditions.

Potential technical problems of the present invention

Most large-scale production methods of antibodies or Fc-containing fusion proteins use Protein A for affinity purification. However, due to the limitations of Protein A in affinity chromatography, there is a need in the art to provide novel Fc-binding proteins with improved properties that specifically bind immunoglobulins to facilitate affinity purification of immunoglobulins. To maximize the value of a chromatography matrix containing Fc-binding proteins, it is desirable to use the affinity ligand matrix multiple times. Between chromatography cycles, a thorough cleaning procedure is required to disinfect and remove residual contaminants from the matrix. In this procedure, it is common practice to apply an alkaline solution with a high concentration of NaOH to the affinity ligand matrix. The wild-type protein A domain cannot withstand such harsh alkaline conditions for a long time and quickly loses its ability to bind immunoglobulins. Therefore, there is an ongoing need in the art to obtain novel alkaline stable proteins capable of binding immunoglobulins.

The present invention provides alkaline-stable immunoglobulin-binding proteins that are particularly suitable for affinity purification of immunoglobulins but overcome the shortcomings of the prior art. In particular, a significant advantage of the alkaline stable Fc-binding proteins of the invention is their improved stability at high pH compared to, for example, wild-type protein A or the parent protein.

The above summary does not necessarily describe all problems addressed by the present invention.

Summary of the invention

A first aspect of the invention is to provide Fc-binding proteins suitable for affinity purification. This is achieved by a basic stable immunoglobulin (Ig) binding protein comprising one or more Fc binding domains, wherein at least one Fc binding domain comprises the amino acid sequence of SEQ ID NO: 1-6 or 21, consisting essentially of Said sequence consists of or consists of said sequence. In one embodiment, the Fc binding protein comprises 2, 3, 4, 5 or 6 Fc binding domains as defined above linked to each other. In some embodiments, the linker connecting the domains is a peptide linker. In preferred embodiments, the Fc-binding protein is conjugated to a solid support.

In some embodiments, the protein is a homo-multimer, and in some embodiments, the protein is a hetero-multimer.

In some embodiments, at least one domain of the protein is a derivative of any one of SEQ ID NOs 1-6 or 21, wherein the derivative has an amino acid sequence that is different from the amino acid sequence relative to the Based on one of SEQ ID NOs: 1-6 or 21 having a deletion of 1, 2, 3 or 4 amino acids within the first 4 amino acids of its N-terminus (positions 1, 2, 3 and/or 4) and/ Or 100% identical to one of SEQ ID NOs: 1-6 or 21, except for a deletion of 1 or 2 amino acids at the C-terminus (position 57 and/or 58).

In some embodiments, the binding capacity of the protein is reduced by less than 15% after incubation in 0.5 M NaOH for at least 5 hours. For example, the protein's binding capacity may be reduced by less than 10% or less than 5% after 6 hours of incubation in 0.5M NaOH.

In a second aspect, the invention relates to an affinity separation matrix comprising the Fc-binding protein of the first aspect.

In a third aspect, the invention relates to the use of the Fc-binding protein of the first aspect or the affinity separation matrix of the second aspect for affinity purification of an immunoglobulin or a protein comprising an Fc sequence of an immunoglobulin.

In a fourth aspect, the invention relates to a method for affinity purifying an immunoglobulin or a protein comprising an Fc sequence of an immunoglobulin, comprising the steps of: (a) providing a liquid containing the immunoglobulin; (b) providing affinity separation A matrix comprising an immobilized Fc-binding protein of the first aspect coupled to the affinity separation matrix; (c) contacting the liquid with the affinity separation matrix, wherein the immunoglobulin is in contact with the immobilized The Fc-binding protein binds; and (d) eluting the immunoglobulin from the matrix to obtain an eluate containing the immunoglobulin. In some embodiments, a wash step can be introduced between steps (c) and (d) of the disclosed methods. In some embodiments of the disclosed uses and methods, the elution of the protein comprising an Fc sequence is greater than or equal to 95% at a pH of 3.5 or higher. For example, at a pH of 3.5 or higher, the protein comprising an Fc sequence elutes greater than or equal to 98%.

In another aspect, the present invention relates to an affinity purification method for a protein comprising an Fc sequence, the method comprising: (a) under conditions allowing the at least one Fc-binding protein to bind to the protein comprising an Fc sequence, comprising: contacting an affinity separation matrix to which at least one Fc-binding protein of the first aspect coupled thereto is contacted with a solution comprising a protein comprising an Fc sequence; and (b) eluting the bound Fc-containing protein from the affinity purification matrix sequence of proteins.

This summary of the invention does not necessarily describe all features of the invention. Other embodiments will become apparent by reviewing the detailed description that follows.

Brief description of the drawings

Figure 1. Analysis of the alkaline stability of different Fc-binding domains immobilized on Sepharose 6B matrix after 6 h of 0.5 M NaOH treatment. The Fc binding domains cs24 (SEQ ID NO:1) and cs26 (SEQ ID NO:2) show significantly improved stability at high pH compared to the parent domain IB24 (SEQ ID NO:17).

Figure 2. Activity analysis of Fc-binding domains immobilized on Praesto ^™ Pure 45 matrix pH 9.5 after 6 hours of incubation in 0.5 M NaOH. Fc binding domains cs24 (SEQ ID NO: 1), cs24a (SEQ ID NO: 3), cs24b (SEQ ID NO: 5), cs26 (SEQ ID NO: 2), cs26a (SEQ ID NO: 4) and cs26b ( SEQ ID NO:6).

Figure 3. Immobilization in Praesto ^TM Pure 85 matrix (Panel A) and Praesto ^TM Pure 45 matrix (Panel A) at pH 9.5 after incubation in 0.5 M NaOH for 6h and 24h (Panel A) and 6h, 24h and 36h (Panel B). Activity analysis of the Fc-binding domain on panel B). Fc binding domains cs24 (SEQ ID NO: 1), cs24a (SEQ ID NO: 3), cs24b (SEQ ID NO: 5), cs26 (SEQ ID NO: 2), cs26a (SEQ ID NO: 4) and cs26b (SEQ ID NO:6) compared to wild-type domain C.

Figure 4. From Fc binding domains cs24 (SEQ ID NO: 1), cs24a (SEQ ID NO: 3), cs24b (SEQ ID NO: 5), cs26 (SEQ ID NO: 2), at pH 3.5 and 2.0 Analysis of cs26a (SEQ ID NO:4) and cs26b (SEQ ID NO:6) eluted polyclonal hlgG. Panel A shows a representative elution test. The step yield for all Fc domains at 3.5 pH elution was greater than 98% (Panel B), far exceeding the elution of Protein A Domain C.

Detailed description of the invention

definition

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the specific methods, protocols, and reagents described herein, as these may vary. It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Preferably, the terms used herein are consistent with "A multilingual glossary of biotechnological terms: (lUPAC Recommendations)", edited by Leuenberger, H.G.W, Nagel, B. and Kolbl, H. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland) consistent with the definitions provided in .

Throughout this specification and the claims that follow, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of stated members unless the context otherwise requires. , an integer or a step or a member, a group of integers or steps, but does not exclude any other member, integer or step or group of members, integers or steps.

As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are used interchangeably and are intended to include the plural form as well. form and falls within each meaning unless the context clearly indicates otherwise. Furthermore, as used herein, "and/or" means and encompasses any and all possible combinations of one or more of the listed items, as well as the lack of combinations when interpreted in the alternative ("or").

As used herein, the term "about" encompasses an expressly recited amount as well as deviations therefrom of ±10%. More preferably, the term "about" includes a deviation of 5%.

Several documents are cited throughout this specification (eg, patents, patent applications, scientific publications, manufacturer's instructions, instructions, GenBank accession number sequence submissions, etc.). Nothing herein shall be construed to mean that the present invention is not entitled to rely on prior invention antedating those disclosures. Some documents cited herein are "incorporated by reference." In the event of a conflict between the definitions or teachings of such incorporated references and the definitions or teachings cited in this specification, the text of this specification shall control.

All sequences mentioned herein are disclosed in the accompanying Sequence Listing, the entire contents and disclosure of which are part of this specification.

In the present description, the term "immunoglobulin-binding protein" is used to describe a protein capable of specifically binding to the Fc region of an immunoglobulin. Due to this specific binding to the Fc region, the immunoglobulin-binding proteins of the invention are capable of binding to entire immunoglobulins, to immunoglobulin fragments comprising the Fc region, to fusion proteins comprising the Fc region of immunoglobulins, and to binding to immunoglobulin fragments comprising the Fc region. Conjugates of the Fc region of immunoglobulins. Although the "immunoglobulin-binding protein" of the present invention shows specific binding to the Fc region of an immunoglobulin, it does not exclude that the "immunoglobulin-binding protein" may additionally bind with reduced affinity to other regions such as immunoglobulins. Fab area combination.

Throughout this specification, the term "immunoglobulin binding protein" is often abbreviated to "Fc-binding protein" or "Fc-binding protein".

In a preferred embodiment of the invention, the Fc binding protein comprises one or more Fc binding domains.

The term "dissociation constant" or " _KD " defines specific binding affinity. As used herein, the term " _KD " (commonly measured in "mol/L" and sometimes abbreviated as "M") is intended to refer to the dissociation equilibrium constant of a specific interaction between a first protein and a second protein. In the context of the present invention, the term _KD is used in particular to describe the binding affinity between an immunoglobulin-binding protein and an immunoglobulin.

A protein of the invention is considered to be a protein if its dissociation constant K _D from an immunoglobulin is at least 1 μM or lower, or preferably 100 nM or lower, more preferably 50 nM or lower, even more preferably 10 nM or lower. Binds to immunoglobulins. For example, all Fc binding domains disclosed in SEQ ID Nos: 1-6 and 21 bind IgG1 with a _KD of less than 1 μM or less.

The term "binding" according to the invention preferably means specific binding. "Specific binding" means that an Fc-binding protein of the invention binds more strongly to an immunoglobulin (or an Fc sequence of an immunoglobulin) for which it is specific than to another non-immunoglobulin target. .

Immunoglobulins as understood herein may include, but are not necessarily limited to, mammalian IgG, such as human IgG1, human IgG2, human IgG4, mouse IgG-1, mouse IgG2A, mouse IgG2 IgG1, rat IgG2C, goat IgG1, goat IgG2 , bovine IgG2, guinea pig IgG, rabbit IgG; human IgM, human IgA; and immunoglobulin fragments comprising the Fc region, fusion proteins comprising the Fc region of the immunoglobulin and conjugates comprising the immunoglobulin Fc region. Notably, the naturally occurring Protein A domains and artificial Fc-binding proteins of the invention do not bind human IgG3.

The terms "protein" and "polypeptide" refer to any linear molecular chain of two or more amino acids linked by peptide bonds and do not refer to a specific length of the product. Thus, "peptide", "protein", "amino acid chain" or any other term used to refer to a chain of two or more amino acids are included within the definition of "polypeptide" and the term "polypeptide" may be used instead Any of these terms or used interchangeably with any of these terms. The term "polypeptide" is also intended to refer to post-translational modifications of the polypeptide (including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, proteolytic cleavage, modification by non-naturally occurring amino acids and similar modifications well known in the art ) product. Therefore, Fc-binding proteins containing two or more protein domains also fall under the definition of the term "protein" or "polypeptide".

The term "alkaline stable" or "alkaline stable" or "caustic stable" or "caustic stable" (herein abbreviated as "cs") means that the Fc binding protein of the invention tolerates alkaline conditions without significant loss of the ability to bind immunoglobulins. One skilled in the art can test for immunity by incubating the Fc-binding protein with sodium hydroxide solution (e.g., as described in the Examples) and subsequently by routine experiments known to those skilled in the art (e.g., by chromatographic methods). Alkaline stability can be easily tested by globulin binding activity.

The Fc-binding proteins of the invention, as well as matrices comprising the Fc-binding proteins of the invention, exhibit "increased" or "improved" alkaline stability, which means that the molecules and matrices incorporating the Fc-binding proteins perform better under alkaline conditions. The protein is stable for an extended period of time relative to the parent Fc-binding protein, that is, does not lose the ability to bind to immunoglobulins or loses the ability to bind to immunoglobulins to a lower extent than the parent Fc-binding protein.

The term "binding activity" refers to the ability of the Fc-binding protein of the invention to bind to immunoglobulins. For example, binding activity can be determined before and/or after alkali treatment. The binding activity of an Fc-binding protein or an Fc-binding protein coupled to a matrix (i.e., an immobilized binding protein) can be determined. The term "artificial" refers to an object that does not occur naturally, that is, the term refers to an object produced or modified by man. For example, a polypeptide or polynucleotide sequence that is produced by humans (eg, in a laboratory by genetic engineering, by shuffling methods, or by chemical reactions, etc.) or intentionally modified is artificial.

As used herein, the term "parent" in the term "parent Fc-binding protein" or "parent Fc-binding domain" refers to an Fc-binding protein that is subsequently modified to produce a variant of the parent protein or domain. Such parent protein or domain may be an artificial Fc binding domain, as disclosed herein as IB24 (SEQ ID NO: 17) or IB26 (SEQ ID NO: 18).

As used herein, the term "conjugate" refers to a molecule comprising or consisting essentially of at least a first protein chemically linked to other substances, such as to a second protein or non-proteinaceous moiety.

The term "substitution" or "amino acid substitution" refers to the exchange of an amino acid at a specific position in the parent polypeptide sequence for another amino acid. Given the known genetic code, as well as recombinant and synthetic DNA technology, skilled artisans can readily construct DNA encoding amino acid variants.

The term "amino acid sequence identity" refers to a quantitative comparison of the identity (or difference) of the amino acid sequences of two or more proteins. "Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the amino acid residues in the sequence that are identical to the reference polypeptide sequence after aligning the sequences and introducing gaps if necessary (to achieve maximum percent sequence identity) percentage of amino acid residues.

To determine sequence identity, the sequence of the query protein is aligned with the sequence of a reference protein. Alignment methods are well known in the art.

The term "fusion" means that the components are connected by peptide bonds, either directly or through a peptide linker.

The term "fusion protein" refers to a protein comprising at least a first protein genetically linked to at least a second protein. Fusion proteins are produced by joining two or more genes that originally encoded separate proteins. Thus, fusion proteins may comprise multimers of the same or different proteins expressed as a single linear polypeptide.

As used herein, the term "linker" in its broadest sense refers to a molecule that covalently links at least two other molecules. In a typical embodiment of the invention, "linker" is understood to be the part that connects the Fc-binding domain to at least one further Fc-binding domain, ie that connects the two protein domains to each other to produce a multimer. In a preferred embodiment, a "linker" is a peptide linker, ie the portion connecting two protein domains is one amino acid or a peptide containing two or more amino acids.

The term "chromatography" refers to a separation technique that uses mobile and stationary phases to separate one type of molecule (eg, immunoglobulins) from other molecules (eg, contaminants) in a sample. The liquid mobile phase contains a mixture of molecules and transports these molecules through or through a stationary phase (such as a solid matrix). Molecules in the mobile phase can be separated due to their differential interaction with the stationary phase.

The term "affinity chromatography" refers to a specific chromatographic mode in which a ligand coupled to a stationary phase interacts with a molecule (i.e., an immunoglobulin) in the mobile phase (sample), i.e., the ligand is Molecules have specific binding affinities. As understood in the context of the present invention, affinity chromatography involves the addition of an immunoglobulin-containing sample to a stationary phase containing a chromatography ligand, such as an Fc-binding protein of the invention.

The terms "solid support" or "solid matrix" are used interchangeably to refer to the stationary phase.

The terms "affinity matrix" or "affinity separation matrix" or "affinity chromatography matrix" as used interchangeably herein refer to a matrix, e.g. a chromatography matrix, to which an affinity ligand is attached, e.g. Invented Fc-binding protein. A ligand (eg, an Fc-binding protein) is capable of specifically binding to a target molecule (eg, an immunoglobulin or Fc-containing protein) to be purified or removed from the mixture.

As used herein, the term "affinity purification" refers to a method of purifying an immunoglobulin or Fc-containing protein from a liquid by binding the immunoglobulin or Fc-containing protein to an Fc-binding protein immobilized in a matrix. Therefore, all other components of the mixture except immunoglobulins or Fc-containing proteins are removed. In an additional step, the bound immunoglobulin or Fc-containing protein can be eluted in a purified form.

Embodiments of the invention

The invention will now be described further. In the following paragraphs, different aspects of the invention are defined in more detail. Unless expressly stated to the contrary, each aspect defined below may be combined with any other aspect. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature indicated as being preferred or advantageous.

In a first aspect, the invention relates to an Fc-binding protein comprising one or more Fc-binding domains, wherein at least one Fc-binding domain comprises the amino acid sequence of SEQ ID NO: 1-6 or 21, consisting essentially of said sequence Consisting of or consisting of said sequence. One advantageous aspect of the disclosed Fc-binding domains and proteins containing the domains is that they remain stable even after alkali treatment, particularly compared to the parent protein and other known Fc-binding proteins. For example, in some embodiments, the disclosed Fc binding domains and proteins comprising the domains may be at least about 15% more stable than Domain C of Protein A, at least about 20%, at least about 25% or at least about 30%. In other words, compared to Protein A Domain C, the disclosed Fc-binding domain has a smaller reduction in binding capacity after incubation with 0.5 M NaOH for ≥5 hours. Thus, in some embodiments, the disclosed Fc protein has less than a 20% reduction in binding capacity upon incubation in 0.5 M NaOH for at least 5 hours (eg, 6 hours). In some embodiments, the disclosed Fc protein may have a decrease in binding capacity of less than about 15%, less than about 10%, or less than about 5% upon incubation in 0.5 M NaOH for at least 5 hours.

All Fc-binding proteins of the invention bind to immunoglobulins with a dissociation constant K _D below 1 μM, or preferably below 100 nM, or even more preferably 10 nM or below. Methods for determining the binding affinity of an Fc-binding protein or domain, i.e. for determining the dissociation constant _K , are known to a person of ordinary skill in the art and may, for example, be selected from the following methods known in the art: surface plasmon based Techniques of bulk resonance (SPR), biolayer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), flow cytometry, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA) ) and enhanced chemiluminescence (ECL). Some methods are further described in the Examples. Typically, the dissociation constant K _D is determined at 20°C, 25°C or 30°C. If not explicitly stated otherwise, K _D values described herein are determined by surface plasmon resonance at 22°C +/-3°C. In one embodiment of the first aspect, the dissociation constant KD of the Fc-binding protein for human IgG1 is in the range of 0.1 nM to 100 nM, preferably 0.1 nM to 10 nM.

As shown in the following examples, it was surprisingly and unexpectedly found that the Fc-binding proteins of the invention still bind to IgG even after prolonged alkaline treatment. In some embodiments, the Fc-binding proteins of the invention exhibit improved alkaline stability compared to the corresponding parent protein. Fc binding was determined by comparing the loss of IgG binding activity of the Fc-binding protein after 6 hours of incubation in 0.5 M NaOH as compared to the loss of IgG binding activity of the corresponding parent protein after 6 hours of incubation in 0.5 M NaOH. Alkaline stability of proteins. Loss of binding activity was determined by comparing binding activity before and after 6 hours of incubation with 0.5 M NaOH.

As shown by the comparative data in Figure 1, the IgG binding activity of cs24 and cs26 was increased by at least about 30% compared to IB24. This is an unexpected and advantageous property of cs24 and cs26 compared to the parent IB24. Figure 2 shows that after 6 hours of incubation in 0.5 M NaOH, all Fc-binding proteins of SEQ ID NO: 1-6 had at least 87.6% binding activity remaining.

In one embodiment of the invention, the Fc-binding protein contains 2, 3, 4, 5 or 6 Fc-binding domains connected to each other, that is, the Fc-binding protein can be a monomer, a dimer, a trimer, a tetramer, or a monomer. body, pentamer or hexamer.

In some embodiments, the domain is selected from SEQ ID NOs: 1-6 and 21. In other embodiments, the domain is a derivative of SEQ ID NO: 1-6 or 21, and additionally wherein each derivative is relative to one of SEQ ID NO: 1-6 or 21 on which it is based has a deletion of 1, 2, 3 or 4 amino acids within the first 4 amino acids of its N-terminus and/or a deletion of 1 or 2 amino acids at the C-terminus (positions 57 and/or 58) (see, e.g. , SEQ ID NO:7-16), each derivative is 100% identical to one of SEQ ID NO:1-6.

The multimers of the present invention are fusion proteins usually produced artificially by recombinant DNA techniques well known to those skilled in the art. The Fc-binding proteins of the invention can be prepared by any of a number of conventional and well-known techniques (such as common organic synthesis strategies, solid-phase assisted synthesis techniques) or by commercially available automated synthesizers.

In some embodiments of the first aspect, the multimer is a homomultimer, eg, the amino acid sequence of all Fc-binding domains of the Fc-binding protein is identical.

In some embodiments of the first aspect, the multimer is a heteromultimer, eg, at least one Fc binding domain has a different amino acid sequence than other Fc binding domains within the Fc binding protein.

In some embodiments of the first aspect, the Fc binding domains are directly linked to each other. In other embodiments, the one or more Fc binding domains are connected to each other through one or more linkers. Preferred among these exemplary embodiments are peptide linkers. This means that the peptide linker is the amino acid sequence connecting the first Fc binding domain to the second Fc binding domain. A peptide linker is connected to the first Fc-binding domain and the second Fc-binding domain via a peptide bond between the C- and N-termini of the domains, thereby creating a single linear polypeptide chain. The length and composition of the linker can vary between at least one and up to about 30 amino acids. More specifically, the peptide linker has 1 to 30 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acids in length. Preferably the amino acid sequence of the peptide linker is stable against caustic conditions and proteases. The linker should not destabilize the conformation of the domain in the Fc-binding protein. Linkers containing small amino acids such as glycine and serine are well known. The linker can be glycine rich (eg, more than 50% of the residues in the linker can be glycine residues). Also preferred are linkers containing additional amino acids. Other embodiments of the invention include linkers consisting of alanine, proline and serine. Other linkers for protein fusion are known in the art and can be used.

In some embodiments of the invention, the Fc binding protein is conjugated to a solid support. In some embodiments of the invention, the Fc-binding protein may also comprise additional amino acid residues at the N- and/or C-terminus, such as, for example, a leader sequence at the N-terminus and/or with or without at the N- or C-terminus. Labeled coupling sequence. In some embodiments, the Fc-binding protein further comprises an attachment site for covalent attachment to a solid phase (matrix). Preferably, the attachment site is specific to provide site-specific attachment of the Fc-binding protein to the solid phase. Specific attachment sites comprise natural amino acids, such as cysteine or lysine, which enable reactive groups with the solid phase or the linker between the solid phase and the protein (e.g. selected from N-hydroxysuccinimide). amine, iodoacetamide, maleimide, epoxy, or olefin groups) for specific chemical reactions. The attachment site may be located directly at the C- or N-terminus of the Fc-binding protein, or there may be a linker, preferably a peptide linker, between the N- or C-terminus and the coupling site. In some embodiments of the invention, the Fc-binding protein may comprise a short N- or C-terminal peptide sequence of 3-20 amino acids, preferably 4-10 amino acids, with a terminal cysteine. The amino acid used for the C-terminal attachment site may preferably be selected from proline, alanine and serine, such as ASPAPSAPSAC (SEQ ID NO: 19), with a single cysteine at the C-terminus for coupling. In another embodiment, the amino acid of the C-terminal attachment site may preferably be selected from glycine and serine, such as GGGSC, with a single cysteine at the C-terminus for coupling.

One advantage of having a C-terminal cysteine is that coupling of Fc-binding proteins can be achieved by reaction of the cysteine thiol with an electrophilic group on the support (creating a thioether bridge coupling). This provides excellent mobility of the coupled protein, which provides increased binding capacity.

In a second aspect, the invention relates to an affinity separation matrix comprising the Fc binding protein of the first aspect.

In a preferred embodiment of the second aspect, the affinity separation matrix is a solid support. The affinity separation matrix comprises at least one Fc-binding protein comprising at least one Fc-binding domain comprising any one of SEQ ID NOs: 1-6 or 21.

The matrix comprising an Fc-binding protein of the invention can be used for separation, for example for chromatographic separation of immunoglobulins and other Fc-containing proteins, such as immunoglobulin variants comprising an Fc region, fusion proteins comprising an Fc region of an immunoglobulin and conjugates comprising the immunoglobulin Fc region. Affinity matrices can be used to isolate immunoglobulins and should maintain Fc-binding properties even under the highly alkaline conditions imposed during cleaning. This cleaning of the substrate is essential for long-term reuse of the substrate.

Solid support matrices for affinity chromatography are known in the art and include, for example, but not limited to, agarose and stabilized agarose derivatives (e.g., Sepharose 6B, Praesto ^™ Pure; PROTEIN A Sepharose Fast Flow), cellulose or cellulose derivatives, controlled pore glass (e.g., /> vA resin), monolithic materials (for example,/> monolith), silica, zirconia (for example, CM zirconia or/> ), titanium oxide or synthetic polymers (e.g. polystyrene such as Poros 50A or Poros/> A resin, polyvinyl ether, polyvinyl alcohol, polyhydroxyalkyl acrylate, polyhydroxyalkyl methacrylate, polyacrylamide, polymethacrylamide, etc.) and hydrogels of various compositions. In certain embodiments, the support includes a polyhydroxy polymer, such as a polysaccharide. Examples of polysaccharides suitable for use as supports include, but are not limited to, agar, agarose, dextran, starch, cellulose, amylopectin, and the like, as well as stabilized variants of these materials.

The solid support matrix may be in the form of any suitable known type. Such solid support matrices for coupling to Fc-binding proteins of the invention may include, for example, one of the following forms: columns, capillaries, particles, membranes, filters, monoliths, fibers, pads, gels, slides Tablets, plates, boxes or any other form commonly used in chromatography and known to those skilled in the art.

In one embodiment, the matrix consists of substantially spherical particles (also called beads, such as Sepharose gel or Agarose beads). Suitable particle sizes may be in the diameter range of 5-500 μm such as 10-100 μm, for example 20-80 μm. Substrates in particulate form can be used as packed beds or in suspended form (including expanded beds).

In an alternative embodiment, the solid support matrix is a membrane, such as a hydrogel membrane. In some embodiments, affinity purification involves a membrane as a matrix to which the Fc-binding protein of the first aspect is covalently bound. The solid support may also be in the form of a membrane in the column.

In some embodiments, affinity purification involves a chromatography column containing a solid support matrix to which the Fc-binding protein of the first aspect is covalently bound.

The Fc-binding proteins of the invention may be attached to a suitable solid support matrix by conventional coupling techniques that utilize, for example, the amino-, thiol- and/or carboxyl groups present in the Fc-binding proteins of the invention. Conjugation can be via nitrogen, oxygen or sulfur atoms of the Fc binding protein. Preferably, the amino acid contained in the N- or C-terminal peptide linker contains said nitrogen, oxygen or sulfur atom.

The Fc-binding protein can be coupled to the support matrix directly or indirectly through spacer elements to provide an appropriate distance between the matrix surface and the Fc-binding protein of the invention, which increases the availability of the Fc-binding protein and facilitates Fc binding of the invention. Chemical coupling of proteins to supports.

Methods of immobilizing protein ligands to solid supports are well known in the art and can be readily performed by those skilled in the art using standard techniques and equipment.

Depending on the Fc-binding protein and the specific conditions, the coupling can be multiple points, for example via several lysines, or single point coupling, for example via a cysteine.

In a third aspect, the invention relates to the Fc-binding protein of the first aspect or the affinity matrix of the second aspect for use in the affinity purification of immunoglobulins or variants thereof (i.e. the use of the Fc-binding protein of the invention for affinity chromatography) the use of. In some embodiments, an Fc-binding protein of the invention is immobilized on a solid support as described in the second aspect of the invention.

In a fourth aspect, the invention relates to a method for affinity purification of a protein comprising an Fc sequence, said method comprising:

(a) providing a solution containing a protein comprising an Fc sequence;

(b) providing thereto an affinity separation matrix comprising at least one Fc-binding protein of the invention;

(c) contacting the affinity separation matrix with the solution under conditions that allow the at least one Fc-binding protein of the invention to specifically bind to a protein comprising an Fc sequence; and (d) extracting from the affinity separation matrix and elution of said protein comprising an Fc sequence in a purification matrix, and

(e) Optionally include washing the affinity matrix between steps (c) and (d).

For the purposes of the disclosed uses and methods, the protein comprising an Fc sequence is an immunoglobulin molecule comprising an Fc sequence or a fragment or derivative thereof, consistent with the definitions provided herein.

Affinity separation matrices suitable for the disclosed uses and methods are those according to the embodiments described above and as known to those skilled in the art.

In some embodiments of the fourth aspect, elution of the immunoglobulin from the matrix in step (d) is accomplished by a change in pH and/or a change in salt concentration. Any suitable solution for elution from the Protein A medium may be used, for example, by a pH 5 or lower solution, or by a pH 11 or higher solution.

In some embodiments, for example, it is preferred to add an additional step (f) for efficient cleaning of the affinity matrix by using an alkaline liquid (eg, pH 13-14). In certain embodiments, the cleaning solution contains 0.1-1.0 M NaOH or KOH, preferably 0.25-0.5 M NaOH or KOH. Due to the high alkaline stability of the Fc-binding proteins of the invention, such highly alkaline solutions can be used for cleaning purposes.

In some embodiments, as steps (a) to (e), optionally the repetitions of (a) to (f) can be repeated at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, At least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times, so the affinity matrix can be reused at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times , at least 70 times, at least 80 times, at least 90 times or at least 100 times.

In general, suitable conditions for carrying out affinity purification methods are known to those skilled in the art. In some embodiments, the disclosed uses or methods comprise affinity purification of the disclosed Fc-binding domains at greater than or equal to 3.5 (e.g., about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5) The pH may provide at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% elution of the Fc-containing protein. In some embodiments, the disclosed Fc binding domains have elution profiles that are superior to Protein A Domain C.

In a fifth aspect, the invention relates to a nucleic acid molecule, preferably an isolated nucleic acid molecule, encoding an Fc-binding protein or Fc-binding domain of any embodiment disclosed above. In one embodiment, the invention relates to a vector comprising said nucleic acid molecule. Vector means any molecule or entity (eg, nucleic acid, plasmid, phage, or virus) that can be used to transfer protein-encoding information into a host cell. In one embodiment, the vector is an expression vector.

In a sixth aspect, the invention relates to an expression system comprising a nucleic acid or vector as disclosed above, such as a prokaryotic host cell, such as E. coli, or a eukaryotic host, such as a yeast (Saccharomyces cerevisiae or Pichia pastoris) or a mammal cells such as CHO cells.

In a seventh aspect, the invention relates to a method for the production of the Fc-binding protein of the first aspect, comprising the steps of: (a) culturing the host cell of the sixth aspect under suitable conditions for expressing the binding protein to obtain the desired The Fc-binding protein; (b) optionally isolating the Fc-binding protein.

Suitable conditions for culturing prokaryotic or eukaryotic hosts are well known to those skilled in the art.

Fc-binding molecules of the invention can be prepared by any of a number of conventional and well-known techniques (such as common organic synthesis strategies, solid phase assisted synthesis techniques) or by commercially available automated synthesizers. On the other hand, they can also be prepared by conventional recombinant techniques alone or in combination with conventional synthetic techniques.

One embodiment of the invention relates to a method for preparing an alkaline stable Fc binding protein comprising at least one Fc binding domain comprising the sequence of any one of SEQ ID NOs: 1-6 or 21, The method includes the following steps: (a) preparing a nucleic acid encoding an Fc-binding protein as defined above; (b) introducing the nucleic acid into an expression vector; (c) introducing the expression vector into a host cell; (d) culturing the the host cell; (e) subjecting the host cell to culture conditions under which the Fc-binding protein is expressed, thereby (e) producing the Fc-binding protein as described above; optionally (f) producing the Fc-binding protein in the isolation step (e) the protein; and (g) optionally conjugating the protein to a solid matrix as described above.

In additional embodiments of the invention, the production of Fc-binding proteins is by cell-free in vitro transcription/translation.

Example

The following examples are provided to further illustrate the invention. However, the present invention is not limited thereto, and the following examples merely show the practicality of the present invention based on the above description.

Example 1. Production of Parental Fc-Binding Proteins of the Invention

The parent protein SEQ ID NO: 17 or SEQ ID NO: 18 was originally produced by a shuffling process of naturally occurring Protein A domains. More specifically, shuffling methods as understood herein are assembly processes that generate artificial amino acid sequences starting from a set of known amino acid sequences that are not identical. The shuffling process includes the following steps: a) providing the sequences of the five naturally occurring Protein A domains E, B, D, A and C and the Protein A variant domain Z; b) aligning the sequences; c) computer Statistical fragmentation is performed to identify subsequences that are recombined, and then d) new artificial sequences of the various fragments are assembled to produce chimeric products, ie novel amino acid sequences. The fragments generated in step c) are of any length, for example, if the fragmented parent sequence is of length n, the fragments are of length 1 to n-1.

The relative positions of the amino acids in the chimeric product are maintained relative to the starting amino acid sequence. At least 90% of positions Q9, Q10, A12, F13, Y14, L17, P20, L22, Q26, R27, F30, 131, Q32, S33, L34, K35, D36, D37, P38, S39, S41, L45, E47 , A48, K50, L51, Q55, A56, P57 are identical between the artificial amino acid sequences of the parent "shuffled" proteins IB24 and IB26 and the naturally occurring Protein A domain or Protein A domain variant, provided that IB24 and Position 4 of IB26 is Q. The overall amino acid sequence of parent protein IB24 or IB26 is artificial because it has no more than about 85% identity to the overall amino acid sequence of any naturally occurring protein A domain or domain Z (e.g., IB24 or IB26 has no more than about 85% identity with the domain B is only 77% identical. After the initial artificial protein has been generated, the protein is further modified by site-specific randomization of the amino acid sequence to further alter the binding properties. Further changes are introduced by site-saturation mutagenesis of individual amino acid residues. Grooming.

The genes for IB24 and IB26 were synthesized and cloned into E. coli expression vectors using standard methods known to the skilled person. DNA sequencing was used to verify the correct sequence of the insert.

To generate multimeric Fc-binding proteins containing more than one binding domain, 2, 3, 4, 5 or 6 Fc-binding domains are genetically fused.

For specific membrane attachment and purification, a short peptide amino acid sequence with a C-terminal Cys (SEQ ID NO:19) and optionally a streptomycin tag (SEQ ID NO:20) is added to the C-terminus of the Fc-binding protein.

Example 2. Mutagenesis of Fc-binding proteins

For site-directed mutagenesis, use according to manufacturer's instructions site-directed Mutagenesis kit (NEB; catalog number E0554S). Several combinations of point mutations were generated by GeneArt ^™ Strings ^™ synthesis (Thermo Fisher Scientific). The Strings DNA fragment corresponds to the purified PCR product and was cloned into a derivative of the pET28a vector. The ligation product was transformed into E. coli XL2-blue cells by electroporation. Individual colonies were screened by PCR to identify constructs containing inserts of the correct size. DNA sequencing was used to verify the correct sequence.

Example 3. Expression of Fc-binding protein

BL21(DE3) competent cells were transformed with an expression plasmid encoding Fc-binding protein. Cells were plated on selection agar plates (kanamycin) and incubated overnight at 37°C. Preculture was inoculated from a single colony into 100 ml of 2xYT medium and grown in a baffled 1L Erlenmeyer flask supplemented with 150 μg/ml kanamycin but without lactose and antifoam on a conventional orbital shaker. Incubate at 37°C, 160 rpm for 16 hours. OD ₆₀₀ reading should be in the range of 6-12. In a 1L thick-walled Erlenmeyer flask supplemented with 150 μg/ml kanamycin from the previous overnight culture (in 400 ml ultra-rich medium (modified H15 medium 2% glucose, 5% yeast extract, 0.89% Glycerol, 0.76% lactose, 250mM MOPS, 202mMTRIS, pH 7.4, antifoam agent SE15) Adjusted starting OD ₆₀₀ to 0.5) Inoculate the main culture. The culture was transferred to a resonant acoustic mixer (RAMbio) and incubated at 37°C at 20xg. Ventilation is facilitated with an Oxy-Pump stopper. Recombinant protein expression is induced by metabolizing glucose and subsequently allowing lactose to enter the cell. OD ₆₀₀ was measured at predetermined time points, and samples adjusted to 5/OD ₆₀₀ were taken out, precipitated and frozen at -20°C. Cells were grown overnight for approximately 24 hours to a final _OD600 of approximately 45-60. To collect biomass, cells were centrifuged at 16,000 x g for 10 min at 20 °C. The pellet was weighed (wet weight) and the pH was measured in the supernatant. Cells were stored at -20°C before treatment.

Example 4: SDS-PAGE analysis of expression and solubility of Fc-binding proteins

Resuspend the sample obtained during the fermentation in 300 μl of extraction buffer (PBS supplemented with 0.2 mg/ml lysozyme, 0.5x BugBuster, 7.5 mM MgSO4, 40 U Benzonase) and mix by incubating in a thermomixer at room temperature. Stir and dissolve at 700 rpm for 15 minutes. Soluble proteins were separated from insoluble proteins by centrifugation (16000xg, 2 min, room temperature). Remove the supernatant (soluble fraction) and resuspend the pellet (insoluble fraction) in an equal amount of urea buffer (8M urea, 0.2M Tris, 2mM EDTA, pH 8.5). Remove 50 µl from the soluble and insoluble fractions and add 12 µl 5x sample buffer along with 5 µl 0.5M DTT. Boil the sample at 95°C for 5 minutes. Finally, 8 μl of those samples were applied to NuPage Novex 4-12% Bis-Tris SDS gels run according to the manufacturer's recommendations and stained with Coomassie. High-level expression of Fc-binding proteins was found under optimized conditions during the selected time period (data not shown). All expressed Fc-binding proteins were soluble to over 95% according to SDS-PAGE.

Example 5: Purification of Fc-binding protein

An Fc-binding protein with a C-terminal StrepTagll (SEQ ID NO:20) was expressed in the soluble fraction of E. coli. Cells were lysed by two freeze/thaw cycles and treated with Strep- The resin undergoes a purification step. To avoid disulfide bond formation, supplement the buffer with 1mMDTT.

Alternatively, Fc-binding proteins with C-terminal StrepTagII were expressed in the soluble fraction of E. coli. Cells were resuspended in cell disruption buffer and lysed by a constant cell disruption system (Unit F8B, Holly Farm Business Park) at 1 kbar for two cycles. Purification steps were performed with Strep-TactinYesin (IBA, Goettingen, Germany) and additional gel filtration (Superdex 75 16/60; GE Healthcare) using the AKTAxpress system (Ge Healthcare) according to the manufacturer's instructions. To avoid disulfide formation, supplement the buffer used for Strep-Tactin purification with 1mM DTT and use citrate buffer (20mM Citrate, 150mM NaCl, pH 6,0) as running buffer for gel filtration liquid.

Example 6. Fc-binding protein binds IgG with high affinity (as determined by surface plasmon resonance experiments)

Equilibrate a CM5 sensor chip (GE Healthcare) with SPR running buffer. Surface-exposed carboxyl groups are activated by passing a mixture of EDC and NHS through, yielding reactive ester groups. Immobilize 700-1500RU of bound ligand (on-ligand) on the flow cell and immobilize the dissociated ligand (off-ligand) on another flow cell. After injection of ethanolamine after ligand immobilization, inject ethanolamine and 10 nM glycine pH 2.0 to remove non-covalently bound Fc-binding proteins. Upon ligand binding, protein analytes accumulate on the surface, increasing the refractive index. This change in refractive index is measured in real time and plotted as response or resonance units (RU) versus time. Analytes were applied to the chip in serial dilutions at the appropriate flow rate (μl/min). After each run, regenerate the chip surface with regeneration buffer and equilibrate with running buffer. Control samples were applied to the matrix. Regeneration and rebalancing were performed as previously described. by using 3000 (GE Healthcare) at 25°C; data were evaluated by BIAevaluation 3.0 software provided by the manufacturer, using the Langmuir 1:1 model (R1=0). The estimated dissociation constants (KD) were normalized for off-target. The binding affinities of SEQ ID NO:1 and SEQ ID NO:2 for human IgG1 (cetuximab), human IgG2 (panitumumab), and human IgG4 (natalizumab) are shown in Table 1.

Table 1. K _D values of Fc-binding proteins of the present invention

Example 7. Alkaline stability of Fc-binding proteins coupled to Sepharose 6B matrix. Purified Fc-binding proteins were coupled to epoxy activation according to the manufacturer's instructions (coupling conditions: pH 9.0 overnight, blocked with ethanolamine for 5 h). matrix (Sepharose 6B, GE; catalog number 17-0480-01). Cetuximab was used as IgG1 sample (5 mg; 1 mg/ml matrix). Cetuximab was administered in saturating amounts to a matrix containing immobilized Fc-binding protein. The matrix was washed with 100 mM glycine buffer, pH 2.5, to elute cetuximab bound to the immobilized IgG binding protein. The concentration of eluted IgG was measured by BLI (quantification with protein AOctet-sensor, cetuximab as standard) to determine the binding activity of the Fc-binding protein. The column was incubated with 0.5M NaOH for 6 h at room temperature (22°C +/-3°C). The immobilized proteins were analyzed for IgG binding activity before and after incubation with 0.5 M NaOH for 6 hours. The IgG binding activity of the immobilized protein before NaOH treatment was defined as 100%.

Figure 1 shows that the activity of the Fc binding proteins SEQ ID NO: 1 and SEQ ID NO: 2 is higher compared to the activity of the parent protein IB24 (parent IB26 is comparable to parent IB24; data not shown). Both Fc binding proteins SEQ ID NO: 1 and SEQ ID NO: 2 showed at least 30% higher IgG binding activity compared to the parent protein IB24 after incubation in 0.5 M NaOH for 6 hours. Therefore, the Fc-binding proteins of the invention display significantly improved stability at high pH compared to the parent protein.

Example 8. Alkaline stability of Fc-binding proteins coupled to agarose-based chromatography beads Praesto ^™ Pure45

The purified Fc-binding protein was coupled to agarose-based chromatography beads (Praesto ^™ Pure45, Purolite; Cat. No. PR01262- 166). Use polyclonal human IgG (Ocatpharm) as IgG sample (concentration 2,2mg/ml). Polyclonal hlgG samples were applied in saturating amounts to a matrix containing immobilized Fc-binding protein. The matrix was washed with 100 mM citrate buffer, pH 2.0, to elute hlgG bound to the immobilized Fc-binding protein. Dynamic binding capacity was determined from the amount of hIgG injected at 10% penetration at a 6 minute residence time. The column was incubated with 0.5M NaOH for 6 h at room temperature (22°C +/-3°C). The immobilized proteins were analyzed for IgG binding activity before and after incubation with 0.5 M NaOH for 6 h. The IgG binding activity of the immobilized protein before NaOH treatment was defined as 100%.

Figure 2 shows that the activity of Fc-binding proteins SEQ ID NO:1-6 is very high even after 6h incubation in 0.5M NaOH (for all Fc-binding proteins SEQ ID NO:1-6 after 6h incubation in 0.5M NaOH , the remaining activity is at least 87.6%). All Fc-binding proteins of the invention display significantly high stability at high pH. Figure 3 further shows the results from Praesto ^™ Pure45 matrix and Praesto ^™ Pure85 matrix.

Example 9. Elution of hlgG from Fc-binding proteins coupled to agarose-based chromatography beads Praesto ^™ Pure45 and/or Pure85

Purified Fc-binding proteins were coupled to agarose-based chromatography beads (Praesto ^™ Pure45 or Pure 5) according to the manufacturer's instructions. Use polyclonal human IgG As an IgG sample (concentration 2.2 mg/ml), load into DBC10%. Polyclonal hlgG samples were applied in saturating amounts to a matrix containing immobilized Fc-binding protein. In the two-step method, the matrix is washed first with 100 mM citrate buffer (pH 3.5) and then with 100 mM citrate buffer (pH 2.0) to elute hlgG bound to the immobilized Fc-binding protein.

As shown in Figure 4, greater than 98% of the bound polyclonal human IgG was eluted at pH 3.5, which was significantly higher than the elution from wild-type Protein A domain C.

sequence list

<110> Navigo Proteins GmbH

<120> Alkaline stable FC-binding protein for affinity chromatography

<130> SP32-3 WO R

<160> 20

<170> PatentIn version 3.5

<210> 1

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> cs24

<400> 1

Ile Ala Ala Gln His Asp Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55

<210> 2

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> cs26

<400> 2

Ile Ala Ala Gln His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55

<210> 3

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> cs24a

<400> 3

Ile Ala Ala Gln His Asp Lys Glu His Gln Ala Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55

<210> 4

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> cs26a

<400> 4

Ile Ala Ala Gln His Asp Lys Asp His Gln Ala Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55

<210> 5

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> cs24b

<400> 5

Ile Ala Ala Gln His Asp Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Arg His Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55

<210> 6

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> cs26b

<400> 6

Ile Ala Ala Gln His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Arg His Asp Pro Ser Val Ser Leu Glu Ile Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55

<210> 7

<211> 53

<212> PRT

<213> Artificial sequence

<220>

<223> cs24 delNC

<400> 7

Gln His Asp Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu

1 5 10 15

Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg

20 25 30

Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu Ala Lys Lys Leu

35 40 45

Asn Asp Ala Gln Ala

50

<210> 8

<211> 53

<212> PRT

<213> Artificial sequence

<220>

<223> cs26 delNC

<400> 8

Gln His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu

1 5 10 15

Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg

20 25 30

Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Ala Glu Ala Lys Lys Leu

35 40 45

Asn Asp Ala Gln Ala

50

<210> 9

<211> 55

<212> PRT

<213> Artificial sequence

<220>

<223> cs24 delN

<400> 9

Ala His Asp Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu

1 5 10 15

Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg

20 25 30

Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu Ala Lys Lys Leu

35 40 45

Asn Asp Ala Gln Ala Pro Lys

50 55

<210> 10

<211> 55

<212> PRT

<213> Artificial sequence

<220>

<223> cs26 delN

<400> 10

Ala His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu

1 5 10 15

Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg

20 25 30

Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Ala Glu Ala Lys Lys Leu

35 40 45

Asn Asp Ala Gln Ala Pro Lys

50 55

<210> 11

<211> 318

<212> PRT

<213> Artificial sequence

<220>

<223> cs24 delNC hexamer

<400> 11

Gln His Asp Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu

1 5 10 15

Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg

20 25 30

Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu Ala Lys Lys Leu

35 40 45

Asn Asp Ala Gln Ala Gln His Asp Lys Glu Gln Gln Ala Ala Phe Tyr

50 55 60

Glu Ile Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe

65 70 75 80

Ile Gln Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Gly

85 90 95

Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Gln His Asp Lys Glu Gln

100 105 110

Gln Ala Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Asp

115 120 125

Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro Ser Val Ser

130 135 140

Leu Glu Ile Leu Gly Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Gln

145 150 155 160

His Asp Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu Pro

165 170 175

Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp

180 185 190

Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu Ala Lys Lys Leu Asn

195 200 205

Asp Ala Gln Ala Gln His Asp Lys Glu Gln Gln Ala Ala Phe Tyr Glu

210 215 220

Ile Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile

225 230 235 240

Gln Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu

245 250 255

Ala Lys Lys Leu Asn Asp Ala Gln Ala Gln His Asp Lys Glu Gln Gln

260 265 270

Ala Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Asp Gln

275 280 285

Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro Ser Val Ser Leu

290 295 300

Glu Ile Leu Gly Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala

305 310 315

<210> 12

<211> 318

<212> PRT

<213> Artificial sequence

<220>

<223> cs26 delNC hexamer

<400> 12

Gln His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu

1 5 10 15

Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg

20 25 30

Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Ala Glu Ala Lys Lys Leu

35 40 45

Asn Asp Ala Gln Ala Gln His Asp Lys Asp Gln Gln Ala Ala Phe Tyr

50 55 60

Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe

65 70 75 80

Ile Gln Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Ala

85 90 95

Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Gln His Asp Lys Asp Gln

100 105 110

Gln Ala Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu

115 120 125

Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro Ser Val Ser

130 135 140

Leu Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Gln

145 150 155 160

His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu Pro

165 170 175

Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp

180 185 190

Asp Pro Ser Val Ser Leu Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn

195 200 205

Asp Ala Gln Ala Gln His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu

210 215 220

Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile

225 230 235 240

Gln Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Ala Glu

245 250 255

Ala Lys Lys Leu Asn Asp Ala Gln Ala Gln His Asp Lys Asp Gln Gln

260 265 270

Ala Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln

275 280 285

Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro Ser Val Ser Leu

290 295 300

Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala

305 310 315

<210> 13

<211> 290

<212> PRT

<213> Artificial sequence

<220>

<223> cs24 pentamer

<400> 13

Ile Ala Ala Gln His Asp Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ile Ala Ala Gln His Asp

50 55 60

Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu

65 70 75 80

Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro

85 90 95

Ser Val Ser Leu Glu Ile Leu Gly Glu Ala Lys Lys Leu Asn Asp Ala

100 105 110

Gln Ala Pro Lys Ile Ala Ala Gln His Asp Lys Glu Gln Gln Ala Ala

115 120 125

Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn

130 135 140

Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile

145 150 155 160

Leu Gly Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ile Ala

165 170 175

Ala Gln His Asp Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile Leu His

180 185 190

Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu

195 200 205

Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu Ala Lys Lys

210 215 220

Leu Asn Asp Ala Gln Ala Pro Lys Ile Ala Ala Gln His Asp Lys Glu

225 230 235 240

Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu

245 250 255

Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro Ser Val

260 265 270

Ser Leu Glu Ile Leu Gly Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala

275 280 285

Pro Lys

290

<210> 14

<211> 290

<212> PRT

<213> Artificial sequence

<220>

<223> cs26 pentamer

<400> 14

Ile Ala Ala Gln His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ile Ala Ala Gln His Asp

50 55 60

Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu

65 70 75 80

Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro

85 90 95

Ser Val Ser Leu Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala

100 105 110

Gln Ala Pro Lys Ile Ala Ala Gln His Asp Lys Asp Gln Gln Ala Ala

115 120 125

Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn

130 135 140

Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile

145 150 155 160

Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ile Ala

165 170 175

Ala Gln His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile Leu His

180 185 190

Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu

195 200 205

Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Ala Glu Ala Lys Lys

210 215 220

Leu Asn Asp Ala Gln Ala Pro Lys Ile Ala Ala Gln His Asp Lys Asp

225 230 235 240

Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu

245 250 255

Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro Ser Val

260 265 270

Ser Leu Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala

275 280 285

Pro Lys

290

<210> 15

<211> 174

<212> PRT

<213> Artificial sequence

<220>

<223> cs24 tetramer

<400> 15

Ile Ala Ala Gln His Asp Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Gly Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ile Ala Ala Gln His Asp

50 55 60

Lys Glu Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu

65 70 75 80

Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro

85 90 95

Ser Val Ser Leu Glu Ile Leu Gly Glu Ala Lys Lys Leu Asn Asp Ala

100 105 110

Gln Ala Pro Lys Ile Ala Ala Gln His Asp Lys Glu Gln Gln Ala Ala

115 120 125

Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn

130 135 140

Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile

145 150 155 160

Leu Gly Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

165 170

<210> 16

<211> 174

<212> PRT

<213> Artificial sequence

<220>

<223> cs26 tetramer

<400> 16

Ile Ala Ala Gln His Asp Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ile Ala Ala Gln His Asp

50 55 60

Lys Asp Gln Gln Ala Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu

65 70 75 80

Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro

85 90 95

Ser Val Ser Leu Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala

100 105 110

Gln Ala Pro Lys Ile Ala Ala Gln His Asp Lys Asp Gln Gln Ala Ala

115 120 125

Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn

130 135 140

Ala Phe Ile Gln Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile

145 150 155 160

Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

165 170

<210> 17

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> IB24 parent

<400> 17

Ala Ala Ala Gln His Asp Lys Glu Gln Gln Ser Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Thr Glu Asp Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Gly Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55

<210> 18

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> IB26 parent

<400> 18

Ala Ala Ala Gln His Asp Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55

<210> 19

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Coupling sequence

<400> 19

Ala Ser Pro Ala Pro Ser Ala Pro Ser Ala Cys Ala Ser

1 5 10

<210> 20

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> streptaq

<400> 20

Trp Ser His Pro Gln Phe Glu Lys

1 5

<210> 21

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> cs26c

<400> 2

Ile Ala Ala Gln His Asp Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Arg Asp Asp Pro Ser Val Ser Leu Glu Ile Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55

Claims (15)

1. An Fc binding domain consisting of the amino acid sequence of SEQ ID NO:1. 2. An Fc-binding protein consisting of 2, 3, 4, 5 or 6 Fc-binding domains of claim 1 connected to each other. 3. The Fc-binding protein of claim 2, wherein the domains are linked to each other directly or via one or more linkers. 4. The Fc-binding protein of claim 3, wherein the linker is a peptide linker. 5. The Fc-binding domain of claim 1 or the Fc-binding protein of any one of claims 2-4, wherein the Fc-binding domain or the Fc-binding protein is incubated in 0.5M NaOH at 22°C for at least 5 hours. with less than 15% reduction in binding capacity. 6. The Fc-binding domain of claim 1 or the Fc-binding protein of any one of claims 2-4, wherein the Fc-binding domain or the Fc-binding protein is conjugated to a solid support. 7. The Fc-binding domain of claim 1 or the Fc-binding protein of any one of claims 2-4, wherein the Fc-binding domain or the Fc-binding protein further comprises for the Fc-binding domain or the Fc-binding protein. An attachment site for site-specific covalent coupling of the Fc-binding protein to a solid support. 8. An affinity separation matrix comprising the Fc-binding domain of claim 1 or the Fc-binding protein of any one of claims 2-7. 9. Use of the Fc binding domain of claim 1 or the Fc binding protein of any one of claims 2 to 7 or the affinity separation matrix of claim 8 for affinity purification of any protein comprising an Fc sequence. 10. The use of claim 9, wherein the elution of the protein comprising an Fc sequence is greater than or equal to 95% at a pH of pH 3.5 or higher. 11. A method for affinity purification of a protein comprising an Fc sequence, the method comprising: (a) providing a solution containing a protein comprising an Fc sequence; (b) providing an affinity separation matrix comprising at least one Fc-binding domain of claim 1 or an Fc-binding protein of any one of claims 2-7 coupled thereto; (c) causing said affinity to specifically bind to a protein comprising an Fc sequence under conditions that allow said at least one Fc-binding domain of claim 1 or the Fc-binding protein of any one of claims 2-7 to specifically bind contacting a separation matrix with the solution; and (d) eluting said protein comprising an Fc sequence from said affinity purification matrix. 12. The method of claim 11, further comprising washing the affinity matrix between steps (c) or (d). 13. The method of claim 11 or 12, wherein the protein comprising an Fc sequence comprises an immunoglobulin molecule. 14. The method of claim 11 or 12, wherein elution of the protein comprising an Fc sequence is greater than or equal to 95% at a pH of pH 3.5 or higher. 15. A method for affinity purification of a protein comprising an Fc sequence, the method comprising: (a) allowing an affinity separation matrix comprising at least one Fc-binding domain of claim 1 or an Fc-binding protein of any one of claims 2-7 coupled thereto to a solution containing a protein comprising an Fc sequence to allow The at least one Fc binding domain or Fc binding protein is contacted under conditions that bind the protein comprising an Fc sequence; and (b) eluting the binding protein comprising an Fc sequence from the affinity purification matrix.